Organ specificity in the circadian control of plant gene expression.

Of the many plant genes whose expressions are controlled by the circadian clock, one of the phosphoenolpyruvate carboxylase kinase genes in soya bean (Glycine max) exhibits the unusual property that its control is organ-specific--it is under circadian control in leaves but not in roots. Preliminary experiments suggest that the same is true for at least one gene in Arabidopsis thaliana. It will be important to define the extent and function of this phenomenon and the underlying mechanism.

How to tell the time: the regulation of phosphoenolpyruvate carboxylase in Crassulacean acid metabolism (CAM) plants.

Crassulacean acid metabolism (CAM) plants exhibit persistent circadian rhythms of CO(2) metabolism. These rhythms are driven by changes in the flux through phosphoenolpyruvate carboxylase, which is regulated by reversible phosphorylation in response to a circadian oscillator. This article reviews progress in our understanding of the circadian expression of phosphoenolpyruvate carboxylase kinase.

Partial purification and characterization of a protein inhibitor of phosphoenolpyruvate carboxylase kinase.

The activity of phosphoenolpyruvate carboxylase (PEPCase) kinase in leaf extracts increased markedly on dilution. This was shown to be caused by the presence of a protein that inhibits the kinase. The inhibitor protein was separated from the kinase and purified partially. It inhibited the kinase reversibly, presumably by a direct interaction; it was neither a protease nor a protein phosphatase. The amounts of kinase and inhibitor in leaves were estimated following separation by hydrophobic chromatography. The amount of inhibitor in the crassulacean acid metabolism plant Kalanchoe fedtschenkoi Hamet et Perrier was sufficient to inhibit the basal level of kinase activity present during the light period and the early stages of the dark period. Similarly, the amount of inhibitor in the C4 plant Zea mays L. was sufficient to inhibit the low amount of kinase activity present in the dark and at moderate light intensity. Analogous to the role of the protein inhibitor of mammalian cyclic AMP-dependent protein kinase, the function of the PEPCase kinase inhibitor may be to inhibit the basal level of kinase present in conditions under which rapid flux through PEPCase is not required.

The regulation of phosphoenolpyruvate carboxylase in CAM plants.

Phosphoenolpyruvate carboxylase catalyses the primary assimilation of CO(2) in Crassulacean acid metabolism plants. It is activated by phosphorylation, and this plays a major role in setting the day-night pattern of metabolism in these plants. The key factor that controls the phosphorylation state of phosphoenolpyruvate carboxylase is the activity of phosphoenolpyruvate carboxylase kinase. Recent work on Crassulacean acid metabolism plants has established this enzyme as a novel protein kinase and has provided new insights into the regulation of protein phosphorylation. Phosphoenolpyruvate carboxylase kinase is controlled by synthesis and degradation in response to a circadian oscillator. The circadian control of phosphoenolpyruvate carboxylase kinase can be overridden by changes in metabolite levels. The primary effect of the circadian oscillator in this system may be at the level of the tonoplast, and changes in kinase expression may be secondary to circadian changes in the concentration of a metabolite, perhaps cytosolic malate.

Phosphoenolpyruvate carboxylase kinase is a novel protein kinase regulated at the level of expression.

Phosphorylation of phosphoenolpyruvate carboxylase plays a key role in the control of plant metabolism. Phosphoenolpyruvate carboxylase kinase is a Ca2+-independent enzyme that is activated by a process involving protein synthesis in response to a range of signals in different plant tissues. The component whose synthesis is required for activation has not previously been identified, nor has the kinase been characterised at a molecular level. We report the cloning of phosphoenolpyruvate carboxylase kinase from the Crassulacean Acid Metabolism plant Kalanchoë fedtschenkoi and the C3 plant Arabidopsis thaliana. Surprisingly, phosphoenolpyruvate carboxylase kinase is a member of the Ca2+/calmodulin-regulated group of protein kinases. However, it lacks the auto-inhibitory region and EF hands of plant Ca2+-dependent protein kinases, explaining its Ca2+-independence. Its sequence is novel in that it comprises only a protein kinase catalytic domain with no regulatory regions; it appears to be the smallest known protein kinase. In K. fedtschenkoi, the abundance of phosphoenolpyruvate carboxylase kinase transcripts increases during leaf development. The transcript level in mature leaves is very low during the photoperiod, reaches a peak in the middle of the dark period and correlates with kinase activity. It exhibits a circadian oscillation in constant conditions. Protein kinases are typically regulated by second messengers, phosphorylation or protein/protein interactions. Phosphoenolpyruvate carboxylase kinase is an exception to this general rule, being controlled only at the level of expression. In K. fedtschenkoi, its expression is controlled both developmentally and by a circadian oscillator.

Evidence for a novel class of microbial 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase in Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa.

The tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthases from Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa have been purified to homogeneity. All three enzymes have a subunit Mr of 54,000. The S. coelicolor DAHP synthase was physically and kinetically characterized and the N-terminal amino acid sequence was obtained. The N-terminal amino acid sequence could not be obtained for the enzymes from S. rimosus and N. crassa, their N-termini apparently being blocked. However, following proteolytic digestion, internal amino acid sequences were obtained from both enzymes. A comparison with the known DAHP synthase sequences indicated that these DAHP synthases are unrelated to other microbial DAHP synthase sequences but are similar to plant DAHP synthases. Up until now, two distinct classes of DAHP synthase have been described, one comprising exclusively enzymes from plants, the other restricted to enzymes from micro-organisms. These studies indicate that the class containing the plant DAHP synthases also contains enzymes from a microbial eukaryote and from several bacteria.

Decarboxylation of Malate in the Crassulacean Acid Metabolism Plant Bryophyllum (Kalanchoe) fedtschenkoi (Role of NAD-Malic Enzyme).

The role of NAD-malic enzyme (NAD-ME) in the Crassulacean acid metabolism plant Bryophyllum (Kalanchoe) fedtschenkoi was investigated using preparations of intact and solubilized mitochondria from fully expanded leaves. Intact, coupled mitochondria isolated during the day or night did not differ in their ability to take up [14C]malic acid from the surrounding medium or to respire using malate or succinate as substrate. However, intact mitochondria isolated from plants during the day decarboxylated added malate to pyruvate significantly faster than mitochondria isolated from plants at night. NAD-ME activity in solubilized mitochondrial extracts showed hysteretic kinetics and was stimulated by a number of activators, including acetyl-coenzyme A, fructose-1,6-bisphosphate, and sulfate ions. In the absence of these effectors, reaction progress curves were nonlinear, with a pronounced acceleration phase. The lag period before a steady-state rate was reached in assays of mitochondrial extracts decreased during the photoperiod and increased slowly during the period of darkness. However, these changes in the kinetic properties of the enzyme could not account for the changes in the rate of decarboxylation of malate by intact mitochondria. Gel-filtration experiments showed that mitochondrial extracts contained three forms of NAD-ME with different molecular weights. The relative proportions of the three forms varied somewhat throughout the light/dark cycle, but this did not account for the changes in the kinetics behavior of the enzyme during the diurnal cycle.

Site-directed mutagenesis of cysteine-195 in isocitrate lyase from Escherichia coli ML308.

Cysteine-195 was previously identified as a probable active site residue in isocitrate lyase (ICL) from Escherichia coli ML308 [Nimmo, Douglas, Kleanthous, Campbell and MacKintosh (1989) Biochem. J. 261, 431-435]. This residue was replaced with serine and alanine residues by site-directed mutagenesis. The mutated genes expressed proteins with low but finite ICL activity, which co-migrated with wild-type ICL on both SDS/ and native PAGE. The mutant proteins were purified and characterized. Fluorimetry and c.d. in both the near- and the far-u.v. regions showed no differences between the mutants and wild-type ICL, indicating that the conformations of the three enzymes were very similar. ICL C195A (Cys-195-->Ala) and C195S (Cys-195-->Ser) showed 8.4-fold and 3.6-fold increases in the Km for isocitrate, while their kcat. values showed 30- and 100-fold decreases respectively. The effect of pH on the kinetic properties of the wild-type and mutant ICLs was investigated. The results showed that the response of the mutant enzymes to pH was simpler than that of the wild-type. For the mutants, ionisation of a group with a pKa of approx. 7.8 affected the Km for isocitrate and kcat.. For the wild-type enzyme, these parameters were affected by the ionization of two or more groups, one of which is presumed to by cysteine-195. The results are consistent with the view that the previously identified group with a pKa of 7.1 whose ionization affects the reaction of ICL by iodoacetate is cysteine-195 itself.

Phosphoenolpyruvate carboxylase from Streptomyces coelicolor A3(2): purification of the enzyme, cloning of the ppc gene and over-expression of the protein in a streptomycete.

Phosphoenolpyruvate carboxylase [PEPC; orthophosphate:oxaloacetate carboxy-lyase (phosphorylating); EC 4.1.1.31] is a major anaplerotic enzyme in the polyketide producer Streptomyces coelicolor A3(2). PEPC was purified from S. coelicolor and the amino-acid sequences of four tryptic peptides were determined. Synthetic oligonucleotides based on the sequences of two of the peptides hybridized to the same bands in various restriction-enzyme digests of S. coelicolor genomic DNA. This hybridization allowed molecular cloning of an 8 kb BamHI fragment of genomic DNA. Partial DNA sequencing of this fragment showed that it could encode amino acid sequences similar to those of PEPC from other microorganisms. A BamHI/PstI fragment was subcloned into the streptomycete high-copy-number plasmid vector pIJ486 and transferred into Streptomyces lividans. The resulting strain over-expressed PEPC activity 21-fold and also over-expressed a protein with a subunit of 100,000 M(r), the same as that of purified S. coelicolor PEPC.

Identification of the histidine residue in Escherichia coli isocitrate lyase that reacts with diethylpyrocarbonate.

Escherichia coli isocitrate lyase was inactivated by diethylpyrocarbonate in a pseudo-first-order process. The enzyme was completely inactivated by modification of a single histidine residue, but slower modification of further residues also occurred. The substrate, isocitrate, and products, glyoxylate and succinate, protected against inactivation by diethylpyrocarbonate but this was not simply due to binding at the active site. Treatment of the inactivated enzyme with hydroxylamine led to only partial recovery of activity. Diethylpyrocarbonate also reacted with sulphydryl groups in isocitrate lyase, as judged by titrations with Nbs2, but this reaction was not responsible for the failure of hydroxylamine to reactivate the enzyme fully. The reactivity of isocitrate lyase to diethylpyrocarbonate declined with pH, following a titration curve for a group of pKa 6.1. Isolation and sequencing of ethoxyformylated peptides showed that the major site of modification by diethylpyrocarbonate was histidine residue 306.

Phosphoglycerate mutase from Streptomyces coelicolor A3(2): purification and characterization of the enzyme and cloning and sequence analysis of the gene.

The enzyme 3-phosphoglycerate mutase was purified 192-fold from Streptomyces coelicolor, and its N-terminal sequence was determined. The enzyme is tetrameric with a subunit Mr of 29,000. It is 2,3-bisphosphoglycerate dependent and inhibited by vanadate. The gene encoding the enzyme was cloned by using a synthetic oligonucleotide probe designed from the N-terminal peptide sequence, and the complete coding sequence was determined. The deduced amino acid sequence is 64% identical to that of the phosphoglycerate mutase of Saccharomyces cerevisiae and has substantial identity to those of other phosphoglycerate mutases.

Circadian rhythms in the activity of a plant protein kinase.

Bryophyllum fedtschenkoi is a Crassulacean acid metabolism plant whose phosphoenolpyruvate carboxylase is regulated by reversible phosphorylation in response to a circadian rhythm. A partially purified protein kinase phosphorylated phosphoenolpyruvate carboxylase in vitro with a stoichiometry approaching one per subunit and caused a concomitant 5- to 10-fold decrease in the sensitivity of the carboxylase to inhibition by malate. The sites phosphorylated in vitro were identical to those phosphorylated in intact tissue. The activity of the protein kinase was controlled in a circadian fashion. During normal diurnal cycles, kinase activity appeared between 4 and 5 h after the onset of darkness and disappeared 2----3 h before the end of darkness. Kinase activity displayed circadian oscillations in constant environmental conditions. The activity of protein phosphatase 2A, which dephosphorylates phosphoenolpyruvate carboxylase, did not oscillate. Treatment of detached leaves with the protein synthesis inhibitors puromycin and cycloheximide blocked the nocturnal appearance of the protein kinase activity, maintained phosphoenolypyruvate carboxylase in the dephosphorylated state and blocked the circadian rhythms of CO2 output that is observed in constant darkness and CO2-free air. The simplest explanation of the data is that there is a circadian rhythm in the synthesis of phosphoenolpyruvate carboxylase kinase.

Illumination increases the phosphorylation state of maize leaf phosphoenolpyruvate carboxylase by causing an increase in the activity of a protein kinase.

Illumination of maize leaves increases the phosphorylation state of phosphoenolpyruvate carboxylase and reduces the sensitivity of the enzyme to feedback inhibition by malate. Red, white and blue light were each found to be equally potent, and the effect of light was blocked by 3(3,4-dichlorophenyl)-1,1-dimethylurea. A phosphoenolpyruvate carboxylase kinase was partially purified from illuminated maize leaves by a three-step procedure. Phosphorylation of phosphoenolpyruvate carboxylase by this protein kinase reached 0.7-0.8 molecules/subunit and correlated with a 3- to 4-fold increase in Ki for malate. The protein kinase was inhibited by L-malate, but was insensitive to a number of other potential regulators. Freshly prepared and desalted extracts of darkened maize leaves contained very little kinase activity, but the activity appeared when leaves were illuminated for 30-60 min before extraction. The catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle, but not that of protein phosphatase 1, could dephosphorylate phosphoenolpyruvate carboxylase. The protein phosphatases 1 and 2A activities of maize leaves were not affected by illumination. It is suggested that the major means by which light stimulates the phosphorylation of phosphoenolpyruvate carboxylase is by an increase in the activity of the protein kinase.

Use of chemical modification in the crystallization of isocitrate lyase from Escherichia coli.

Two different crystal forms of isocitrate lyase (ICL) from Escherichia coli have been grown following the chemical modification of the enzyme by either 3-bromopyruvate or ethyl mercuri thiosalicylate (EMTS), contrasting strongly with difficulties in obtaining ordered crystals of the native enzyme. Both crystal forms are obtained using the hanging drop method of vapour diffusion with ammonium sulphate as the precipitant. The crystals diffract well and X-ray photographs of the crystals have established that they are in space groups C222(1) and P3(1) (or its enantiomorph P3(2), respectively. Considerations of the values of Vm and measurements on the crystal density indicate that the asymmetric unit of both crystals contains four subunits.

The purification and characterization of 3-dehydroquinase from Streptomyces coelicolor.

The enzyme 3-dehydroquinase was purified over 4000-fold to homogeneity from Streptomyces coelicolor. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of SDS was 16,000. The native Mr estimated by gel filtration on a Superose 6 column was 209,000, indicating that the enzyme is a large oligomer. The enzyme was found to be extremely thermostable. This stability, along with the structural and kinetic properties of the enzyme, suggest that it is very similar to the quinate-inducible 3-dehydroquinase found in Neurospora crassa and Aspergillus nidulans. This similarity was confirmed by direct N-terminal sequencing.

Regulation of phosphoenolpyruvate carboxylase: an example of signal transduction via protein phosphorylation in higher plants.

There is now good evidence that the malate sensitivity of PEPc is regulated by phosphorylation/dephosphorylation in the leaf tissue of C4 and CAM plants. This statement is based on the assessment of the phosphorylation state of PEPc in [32P]-labeled intact tissue by immunoprecipitation and the correlation between phosphorylation state and malate sensitivity that has been observed during incubation of purified PEPc in vitro with protein kinases or protein phosphatases. The phosphorylation of PEPc in the CAM plant B. fedtschenkoi is controlled by an endogenous rhythm whereas that of PEPc in the C4 plant maize is triggered directly by light. In neither case has the mechanism of signal transduction been identified. It is hoped that further work on the protein kinases and protein phosphatases involved will reveal the nature of the signalling systems. Preliminary work suggests that plant protein phosphatases are very similar to their mammalian counterparts. It is also noteworthy that higher plant genes very similar to the genes encoding the cyclic nucleotide-dependent protein kinases and the protein kinase C family have recently been identified. It is interesting to speculate that the protein kinases and phosphatases involved in signal transduction systems in plants may prove to be closely related to well-studied mammalian enzymes.

Studies of the phosphorylation of Escherichia coli isocitrate dehydrogenase. Recognition of the enzyme by isocitrate dehydrogenase kinase/phosphatase and effects of phosphorylation on its structure and properties.

Escherichia coli isocitrate dehydrogenase is completely inactivated by phosphorylation of a single serine residue per subunit. We have examined the conformations of the active and phosphorylated forms of the enzyme using circular dichroism spectroscopy. The results support the view that phosphorylation prevents the binding of NADP, probably by direct blocking of the coenzyme-binding site. Labelling studies suggest that an arginine residue at the coenzyme-binding site may be close to the phosphorylatable serine residue. The phosphorylation of isocitrate dehydrogenase is thus unusual in that it occurs at the active site of the enzyme. We therefore investigated the recognition of isocitrate dehydrogenase by isocitrate dehydrogenase kinase/phosphatase. The kinase activity of this enzyme can phosphorylate intact isocitrate dehydrogenase but not proteolytic fragments derived from it, nor a synthetic peptide corresponding to the sequence round the phosphorylation site.

Insulin stimulates the tyrosyl phosphorylation and activation of the 52 kDa peripheral plasma-membrane cyclic AMP phosphodiesterase in intact hepatocytes.

The 52 kDa subunit of the peripheral-plasma-membrane insulin-stimulated high-affinity cyclic AMP phosphodiesterase can be specifically detected by the antibody PM1 by Western-blotting procedures and also can be immunoprecipitated from a hepatocyte extract. PM1-mediated immunoprecipitation from hepatocyte extracts showed that insulin treatment of intact 32P-labelled hepatocytes caused the rapid phosphorylation of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. Phosphoamino acid analysis and the use of a phosphotyrosine-specific antibody indicated that phosphorylation occurred on tyrosyl residue(s) of this phosphodiesterase. Prior treatment of hepatocytes with glucagon (10 nM) completely blocked the insulin-mediated tyrosyl phosphorylation of this 52 kDa protein, as detected with both the PM1 and the anti-phosphotyrosine antibodies. Treatment of hepatocytes with glucagon alone did not increase the phosphorylation state of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. The specific anti-phosphotyrosine antibody also detected the insulin-stimulated phosphorylation of proteins of 180 kDa, 95 kDa and 39 kDa. Prior treatment of hepatocytes with glucagon decreased the ability of insulin to phosphorylate the 180 kDa and 39 kDa species, but not the 95 kDa species.

Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase.

A method was developed for the purification of phosphoenolpyruvate carboxylase from darkened maize leaves so that the enzyme retained its sensitivity to inhibition by malate. The procedure depended on the prevention of proteolysis by the inclusion of chymostatin in the buffers used during the purification. The purified enzyme was indistinguishable from that in crude extracts as judged by native polyacrylamide-gel electrophoresis. SDS/polyacrylamide-gel electrophoresis followed by immunoblotting, and Superose 6 gel filtration. Gel-filtration studies showed that the purified enzyme and the enzyme in extracts of darkened or illuminated leaves showed a concentration-dependent dissociation of tetrameric into dimeric forms. Purified phosphoenolpyruvate carboxylase and enzyme in crude extracts from darkened leaves were equally sensitive to inhibition by malate (Ki approx. 0.30 mM) under conditions where it existed in the tetrameric or dimeric forms, but the enzyme in crude extracts from illuminated leaves was less sensitive to malate inhibition (Ki approx. 0.95 mM) whether it was present as a tetramer or as a dimer. It is concluded that changes in the oligomerization state of phosphoenolpyruvate carboxylase are not directly involved in its regulation by light.